The chemical state of the SH groups of proteins or other oxidation-sensitive structures often has an effect on the identity, activity, or effective concentration of proteins. Identity means the respective folding of a protein. Activity should be understood as meaning enzyme activity. The effective concentration of a protein should be the proportion of the protein in a solution, which is correctly folded with respect to the biological in vivo function.
It is known from the literature that structure-modifying oxidations of proteins can be suppressed by the use of thiol reagents such as, for example, 2-mercaptoethanol or cysteine.
For example, D-amino acid oxidase (DAO) can be stabilized by thiols. The flavoprotein DAO catalyzes the stereospecific deamination of D-amino acids to the corresponding α-ketoacids and ammonium (P. Golini et al., Enzyme and Microbial Technology 17:324-329 (1995)). However, the addition of thiols such as, for example, cysteine can also reduce the activity of proteins. This effect can also be explained by the presence of cysteine radicals. An example of this is aminoacylase. Aminoacylase is a dimeric enzyme having one Zn2+ atom per subunit. Each subunit of the enzyme contains 2 cysteine SH groups and 2 disulfide bonds. The chemical modification of the SH groups, such as the breaking of the disulfide bonds, can lead to an inactivation of the enzyme. It was possible to show that, by the addition of 2-mercaptoethanol, the activity of the aminoacylase is reduced, whereas after removal of the 2-mercaptoethanol by dialysis or gel filtration the original enzyme activity can be almost completely restored (W. Kördel and F. Schneider, Biochem. Biophys. Acta 445:446-457 (1976)).
For cysteine and some derivatives, it was possible for certain preparation forms and specific applications to demonstrate an antibacterial, antiviral or antifungal activity to a certain extent. Thus, for example, it was possible to show that the addition of cysteine is suitable to a certain extent as protection against the spoilage of foods (U.S. Pat. No. 4,937,085).
With the aid of genetic engineering processes, it is possible to synthesize recombinant proteins, such as insulin or its precursors, and also insulin derivatives which have amino acid compositions differing from the derived gene sequence (e.g., human), in genetically modified microorganisms, such as the bacterium Escherichia coli. 
Recombinant synthesis in microorganisms is carried out with the aid of expression vectors. These expression vectors consist of a vector plasmid containing a control sequence for the replication of the plasmid and a selection gene (inter alia, antibiotic resistance gene, metabolic marker). The coding region of the gene for the protein of interest (e.g., insulin) may be inserted under the control of a promoter that is active in the chosen microorganism. For example, if one chooses E. coli, the lac promoter may be used to control expression of the protein of interest.
A process for the production of recombinant proteins (e.g., insulin or insulin derivatives) with the cooperation of genetically modified microorganisms is composed of a series of process steps, which intermesh with each other and must be coordinated with one another.
Thus, for example, a process for the production of human insulin in E. coli can be constructed from the following process steps:
Fermentation of the microorganisms; cell separation; cell disruption; isolation and intermediate storage of the fusion protein with cysteine; refolding into the native spatial structure including formation of the correct disulfide bridges and subsequent separation of foreign proteins not containing material of value; enzymatic cleavage to the arginylinsulin; basic purification of the aqueous protein solution; 1st chromatographic purification; enzymatic cleavage to human insulin; 2nd chromatographic purification; high purification by means of HPLC; recrystallization; and drying.
The large number of individual steps carried out, as a rule, leads to a considerable loss in total yield, because losses in the specific yield of each individual process step are unavoidable.
By optimizing these intermediate steps, the total yield can be improved. There is considerable interest in such processes in order to improve economical utilization of the resources employed and to decrease environmental pollution.
For example, EP 0906918 describes an improved process for the production of a precursor of insulin or insulin derivatives having correctly linked cystine bridges in the presence of cysteine or cysteine hydrochloride and of a chaotropic auxiliary.
Insulin derivatives are derivatives of naturally occurring insulins, such as human insulin or animal insulins. These insulin derivatives differ from the naturally occurring insulin by the deletion, substitution, and/or addition of at least one genetically encodable amino acid residue in the naturally occurring insulin.
During the storage of proteins, a decrease in the effective concentration usually occurs.
The storage of production products between individual process steps may be necessary for various reasons. For example, a subsequent industrial processing step may not be able to accept the total amount of product of the preceding process step, thus requiring storage of a portion of the previous product.
The time needed for intermediate storage may be of differing length. Owing to capacity, the need to coordinate industrial units, the required delivery of more chemicals or appliances, or other reasons, it may be necessary to extend the intermediate storage to several weeks.
One effective way of increasing the yield of the final product is to reduce the loss of active protein that may occur during the unavoidable intermediate storage of products from individual process steps before further processing takes place.
The use of cysteine or its derivatives for the control of the potential loss in yield of active protein during intermediate storage of production products has not been described until how. These products can differ in composition, as well as in form, including biological components, for example, with participation of enzyme catalysts or genetically modified microorganisms. Such processes are used, for example, in the preparation of insulin.
Depending on the process step, the production products to be intermediately stored can consist of, inter alia, different amounts of complex macromolecules of a biological nature (e.g., proteins, DNA, fats), microorganisms, buffer substances, and starting materials.
The production process, as a rule, is aimed at the preparation of a substance that is as uniform as possible, e.g., the production of insulin from a genetically modified microorganism. If intermediate products have to be stored, e.g., in the preparation of insulin, loss of effective concentration of this protein regularly occurs.
Storage of a protein should be understood as meaning any storage of the protein, regardless of the volumetric amount in which the protein is present, the time period of the storage, or the temperature conditions under which the storage takes place. The storage of proteins normally takes place in aqueous solutions.
An aqueous solution of a protein may contain constituents of nutrient media for the culture of microorganisms in defined form or as a complete media containing, in particular, carbon sources or nitrogen sources, amino acids, inorganic salts, and trace elements. The aqueous solution may also contain buffer components of different chemical buffer types, as well as macromolecules of biological origin, such as DNA or fats. The aqueous solution may additionally contain organic or inorganic compounds such as, for example, sodium dodecylsulfate (“SDS”) or potassium acetate, and also proportions of solvents of differing priority, such as methanol or petroleum ether.